KR20130018711A - Tricyclic indole derivatives as pbr ligands - Google Patents

Abstract

The present invention provides a PET tracer with improved properties for imaging peripheral benzodiazepine receptors (PBR) compared to known PET tracers. The present invention also provides precursor compounds useful for the production of the PET tracer of the present invention, and methods of making the precursor compound and the PET tracer. The present invention also provides a radiopharmaceutical composition comprising the PET tracer of the present invention. Also provided are methods of using PET tracers and radiopharmaceutical compositions.

Description

Tricyclic indole derivatives as PPR ligands {TRICYCLIC INDOLE DERIVATIVES AS PBR LIGANDS}

The present invention relates to in vivo imaging of peripheral benzodiazepine receptor (PBR) and in particular to positron-emitting tomography (PET) imaging. Provided is an indole-based PET tracer that binds to PBR with high affinity, has good brain uptake after administration, and selectively binds to PBR excellently. The present invention also provides precursor compounds useful for the synthesis of the PET tracer of the present invention as well as methods of synthesizing such precursor compounds. Another aspect of the present invention includes a method for synthesizing a PET tracer of the present invention comprising a use of the precursor compound of the present invention, a kit for performing the method, and a cassette for performing an automated version of the method. In addition, the present invention provides a radiopharmaceutical composition comprising the PET tracer of the present invention as well as a method of using the PET tracer.

Peripheral benzodiazepine receptors (PBRs) are known to be localized primarily in peripheral tissues and glial cells, but their physiological function remains unclear. PBR is also referred to as the transponder protein (TSPO). Under the cell, PBR is known to localize on the mitochondrial outer membrane, indicating a potential role in mitochondrial function regulation and the immune system. Moreover, PBR has been considered to be involved in cell proliferation, steroidogenesis, calcium flow and cellular respiration.

Abnormal PBR expression has been described in multiple sclerosis (Banati et al. 2001 Neuroreport; 12 (16): 3439-42, Debruyne et al. 2002 Acta Neurol Belg; 102 (3): 127-35), Rasmussen encephalitis ( Banati et al. 1999 Neurology; 53 (9): 2199-203), cerebrovascular disease (Goerres et al. 2001 Am J Roentgenol; 176 (4): 1016-8), herpes encephalitis (document [ Central nervous system (CNS) including Cagnin et al. 2001 Brain; 124 (Pt 10): 2014-27] and AIDS-associated dementia (Hammoud et al. 2005 J Neurovirol; 11 (4): 346-55) Is associated with an inflammatory disease state.

Also in the CNS, connectivity with PBR is associated with degenerative diseases such as Parkinson's disease (Gerhard et al. 2006 Neurobiol Dis; 21 (2): 404-12), Ouchi et al. 2005 Ann Neurol; 57 (2): 161-2]), cortical basal degeneration (Gerhard et al. 2004 Mov Disord; 19 (10): 1221-6), progressive nuclear paralysis (Gerhard et al. 2006 Neurobiol Dis; 21 (2): 404-12]), multisystem atrophy (Gerhard et al. 2003 Neurology; 61 (5): 686-9), Huntington's disease (Pavese et al. 2006 Neurology; 66 (11): 1638-43 Tai et al. 2007 Brain Res Bull; 72 (2-3): 148-51), Amyotrophic lateral sclerosis (Turner et al. 2004 Neurobiol Dis; 15 (3): 601-9) And Alzheimer's disease (Cagnin et al. 2001 Lancet; 358 (9283): 766, Yasuno et al. 2008 Biol Psychiatry; 64 (10): 835-41).

Ischemic stroke (Gerhard et al. 2005 Neuroimage; 24 (2): 591-5), peripheral nerve injury (Banati et al. 2001 Neuroreport; 12 (16): 3439-42), epilepsy (document A number of CNS ischemic conditions, including Sauvageau 2002 Metab Brain Dis; 17 (1): 3-11, Kumar et al. 2008 Pediatr Neurol; 38 (6), have been shown to be associated with abnormal PBR expression. According to the reported increase in PBR expression in animal models of traumatic brain injury (Venneti et al. 2007 Exp Neurol; 207 (1): 118-27), PBR is a biomarker that measures the extent of injury in traumatic brain injury. (Toyama et al. 2008 Ann Nucl Med; 22 (5): 417-24). Interestingly, acute stress is associated with increased PBR expression in the brain, while chronic stress is associated with downregulation of PBR (Lehmann et al. 1999 Brain Res; 851 (1-2): 141). -7]). Delineation of glioma borders has been reported to be possible using [ ¹¹ C] PK11195 to image PBR (Junck et al. 1989 Ann Neurol; 26 (6): 752-8). PBR may also be associated with neuropathic pain (Tsuda et al. Have observed activated microglia in subjects with neuropathic pain (2005 TINS 28 (2) pp101-7) ).

In the periphery, PBR expression is associated with lung inflammation (Branley et al. 2008 Nucl. Med. Biol; 35 (8): 901-9), chronic obstructive pulmonary disease and asthma (Jones et al. 2003 Eur Respir J). 21 (4): 567-73), inflammatory bowel disease (Ostuni et al. 2010 Inflamm Bowel Dis; 16 (9): 1476-1487), rheumatoid arthritis (van der Laken et al. 2008 Arthritis Rheum; 58 (11): 3350-5]), primary fibromyalgia (Faggioli et al. 2004 Rheumatology; 43 (10): 1224-1225), nerve injury (Durrenberger et al. 2004 J Peripher) Nerv Syst; 9 (1): 15-25]), atherosclerosis (Fujimura et al. 2008 Atherosclerosis; 201 (1): 108-111), colon cancer, prostate cancer and breast cancer (Deane et al. 2007 Mol Cancer Res; 5 (4): 341-9, Miettinen et al. 1995 Cancer Res; 55 (12): 2691-5, Han et al. 2003 J Recept Signal Transduct Res; 23 (2) -3): 225-38]), kidney inflammation (Tam et al. 1999 Nephrol Dial Transplant; 14 (7): 1658-66), Cook et al. 1999 Kidney Int; 55 (4): 1319- 26]) and ischemia -Reperfusion injury (Zhang et al. 2006 J Am Coll Surg; 203 (3): 353-64).

Positron emission tomography (PET) imaging with the PBR selective ligand (R)-[ ¹¹ C] PK11195 provides a general indication of central nervous system (CNS) inflammation. However, (R)-[ ¹¹ C] PK11195 is known to have high protein binding and low specific versus non-specific binding. Moreover, the role of its radiolabeled metabolites is unknown and quantification of binding requires complex modeling. As a result, efforts have been made to develop in vivo imaging agents for PBR that do not suffer from this problem. One such in vivo imaging agent is the tetracyclic indole derivative described in WO 2010/109007, which has good affinity for PBR, good brain uptake and specificity for PBR, and a high radioactivity ratio in the brain 60 minutes after injection. Represents a parent in vivo imaging agent. WO 2010/109007 discloses that particularly preferred in vivo imaging agents are the following ¹⁸ F-labeled compounds:

There is a need for further improved in vivo imaging agents for imaging PBR.

SUMMARY OF THE INVENTION [

The present invention maintains the advantageous properties of known tricyclic indole PET tracers and also provides a PET tracer having a number of improved properties. Compared with known tricyclic PET tracers, the PET tracers of the present invention have improved binding affinity for PBR, slightly improved metabolic profiles with high activity ratios at 60 minutes post-injection showing activity in the brain, and PBR- It was demonstrated that the specific binding to the expression tissue was significantly improved. The present invention also provides precursor compounds useful for the production of the PET tracer of the present invention, as well as methods of making the precursor compound and the PET tracer. The present invention also provides a radiopharmaceutical composition comprising the PET tracer of the present invention. Also provided are methods of using PET tracers and radiopharmaceutical compositions.

<Detailed Description of the Invention>

PET tracer

In one aspect, the present invention provides a positron-emitting tomography (PET) tracer having the following chemical structure:

Wherein the chiral center has the (S) configuration.

A "PET tracer" is a chemical compound comprising a positron-emitting isotope, wherein the chemical compound is designed to target a particular physiology or pathophysiology in a biological system. The presence of positron-emitting isotopes allows the PET tracer to be detected after administration to a biological system, thus facilitating detection of specific physiology or pathophysiology.

The PET tracer of the present invention has been shown to have an affinity nearly five times greater than the affinity of its alternative enantiomers, and nearly twice that of the racemic mixture. It has also been found that the PET tracer of the present invention works better in vivo compared to its alternative enantiomers. The PET tracer of the present invention also works better in vivo compared to the racemic mixture comprising the PET tracer and its alternative enantiomers.

Alternative enantiomers of the PET tracer of the present invention have the following structure:

Wherein the chiral center has (R) configuration.

The term “enantiomer” as used in the present invention refers to one of two enantiomeric forms of enantiomerically pure compounds, ie optically active molecules. Thus, enantiomers are compounds with only one chirality, and the term “chirality” refers to the properties of a compound having an mirror image that has no internal symmetry and is not superimposable. The most frequent cause of chirality in chemical compounds is the presence of asymmetric carbon atoms. An equimolar mixture of a pair of enantiomers is referred to herein as "racemate" or as "racemic mixture".

In the biodistribution experiments described in Example 9, the PET tracer of the present invention shows improved binding of the brain to PBR-rich tissue (ie olfactory bulb) compared to both its alternative enantiomers and racemic mixtures. The results of the in vivo blocking experiments described in Example 11 confirm this finding. The results of the experiments described in Example 10 demonstrated that at 60 minutes the activity in the brain due to the parent compound was improved for the PET tracer of the invention compared to the racemic mixture of the PET tracer and its alternative enantiomers. In addition, in the autoradiography experiment described in Example 12, it was found that the PET tracer of the present invention had a higher selective binding to the neuroinflammatory region compared to the racemic mixture comprising the PET tracer and its alternative enantiomers. Proven. In addition, it was found that the PET tracer of the present invention did not racemize after incubation in human plasma or in the rat S9 fraction for an extended period of time as described in Example 8 below.

Precursor compound

PET tracers of the present invention can be prepared via suitable precursor compounds. Thus, in another aspect, the present invention provides a precursor compound for the preparation of the PET tracer of the present invention, wherein the precursor compound has the formula

<Formula I>

Wherein R ¹ is hydroxyl or a leaving group.

“Precursor compound” includes non-radioactive derivatives of the PET tracer of the invention, in which a chemical reaction with a convenient chemical form of ¹⁸ F occurs site-specific; Can be performed with a minimum number of steps (ideally a single step); Without the need for significant purification (ideally without further purification), it is designed to obtain the PET tracer of the present invention. Such precursor compounds are synthetic compounds and can conveniently be obtained with good chemical purity.

"Leaving group" in the context of the present invention refers to an atom or group of atoms that is replaced as a stable species during a substitution or replacement radiofluorination reaction. Examples of suitable leaving groups are halogen chloro, bromo and iodo, and sulfonate ester mesylate, tosylate, nosylate and triflate. Preferably, the leaving group is selected from mesylate, tosylate and triflate, most preferably mesylate. When the leaving group is mesylate, the precursor compound is referred to herein as "precursor compound 1".

Preparation of Precursor Compounds

Precursor compounds of the invention can be obtained by a variety of different routes, each of which forms a separate aspect of the invention.

Therefore,

(i) providing a racemic mixture of a precursor compound of formula I and a compound of formula II as defined herein:

&Lt;

(Wherein, R ² is for R ¹ as defined above, R ¹ and R ² Is the same);

(ii) separating the precursor compound of formula (I) from the compound of formula (II)

It provides a first method for preparing a precursor compound of formula (I) as defined herein.

"Separating" the precursor compound of formula (I) from the compound of formula (II) is carried out by enantiomeric separation techniques. Suitable enantiomeric separation techniques include high performance liquid chromatography (HPLC), supercritical fluid chromatography (SFC), and simulated layer chromatography (SBC). A detailed evaluation of the various techniques that can be applied for enantiomeric separation can be found in "Chiral Separation Techniques: a Practical Approach" (2007 Wiley; Subramanian, Ed.).

<Reaction Scheme 1>

Scheme 1 illustrates one method for obtaining a racemic mixture of a precursor compound of formula (I) and a compound of formula (II). In Scheme 1, PG represents a hydroxyl protecting group; LG represents a leaving group as defined herein; OTs represent leaving tosylate; IPA stands for isopropyl alcohol. Compound g is a precursor compound of the invention wherein R ¹ is hydroxyl. Suitable hydroxyl protecting groups are well known in the art and include acetyl, benzyl, benzoyl, silyl ethers, alkyl ethers and alkoxymethyl ethers. Such protecting groups are discussed in more detail in Theorodora W. Greene and Peter GM Wuts, "Protective Groups in Organic Synthesis" (Fourth Edition, John Wiley & Sons, 2007). Preferred hydroxyl protecting groups in the context of the invention are benzyl. Scheme 1 is described by Napper et al. (J Med Chem 2005; 48: 8045-54) and Davies et al. (J Med Chem 1998; 41: 451-467).

<Reaction Scheme 2>

<Scheme 2 (continued)>

An alternative method for obtaining a racemic mixture of a precursor compound of formula (I) and a compound of formula (II) is described in Scheme 2 above. In Scheme 2, PG is a hydroxyl protecting group as defined above, THF is tetrahydrofuran and KHMDS is potassium bis (trimethylsilyl) amide. From compound f, scheme 2 proceeds from compound f as described in scheme 1 to yield the resulting racemic mixture. Scheme 2 is based on the method disclosed in WO 2003/014082. In this synthetic route, chlorine in the lower position of the left ring causes cyclization to occur in only one direction. However, when the inventors directly applied the teachings of WO 2003/014082 to obtain racemic mixtures of precursor compounds of formula (I) and compounds of formula (II), the yields were low. This problem was overcome by changing the solvent system used in the cyclization step. In WO 2003/014082 the cyclization step was carried out in toluene, but the inventors have found that an optimum yield is obtained when using diethyl ether instead of toluene. The product of the cyclization step was dissolved in diethyl ether, while the uncyclized starting compound was not. Thus, the uncyclized starting compound remains with ZnCl _{2 at} the bottom of the reaction vessel and the cyclized product migrates into diethyl ether at the top of the reaction vessel.

The second method for obtaining the precursor compound of formula (I) is

(i) providing a compound of formula III

<Formula III>

Wherein PG ¹ is a hydroxyl protecting group;

(ii) converting the compound of Formula III into its corresponding acid chloride;

(iii) reacting the acid chloride obtained in step (ii) with diethylamide to obtain a compound of formula

(IV)

Wherein PG ² is a hydroxyl protecting group and is the same as PG ¹ ;

(iv) deprotecting the compound of formula IV obtained in step (iii) to obtain a hydroxyl derivative;

(v) optionally, adding a leaving group as defined herein

It includes.

Both steps (iv) and (v) produce a precursor compound of formula (I) as defined herein, wherein in step (iv) R ¹ of formula I is hydroxyl and in step (v) R of formula I ¹ is a leaving group.

Step (ii) of “converting” the compound of formula III to acid chloride may be carried out using a reagent selected from oxalyl chloride, thionyl chloride, phosphorus trichloride or phosphorus pentachloride. Oxalyl chloride is preferred.

The “deprotection” step refers to the removal of hydroxyl protecting groups and can be performed by means well known to those skilled in the art. The hydroxyl protecting group PG ¹ is as defined above for PG in Scheme 1. The method used is modified for a particular hydroxyl protecting group. Typical strategies for the removal of hydroxyl protecting groups include hydrolysis, and treatment with acids or bases.

"Adding" the leaving group may be carried out by reacting the compound g of Scheme 1 with a halide derivative of the desired leaving group under suitable reaction conditions. For example, to add mesylate, compound g of Scheme 1 may be reacted with methanesulfonyl chloride in the presence of a base, for example an amine base such as triethylamine.

In step (i) of the second method for obtaining the precursor compound of formula (I), the compound of formula (III) can be provided by various routes. E.g,

(a) providing a racemic mixture of a compound of formula (V) with a compound of formula (VI)

(V)

&Lt; Formula (VI)

(Wherein,

R ¹ is chiral alcohol;

PG ³ and PG ⁴ are the same, each being a hydroxyl protecting group);

(b) separating the compound of formula V from the compound of formula VI;

(c) using acidic conditions to remove R ¹ from the isolated compound of formula V, thereby producing a compound of formula III

Follow the method including the.

The term "racemic mixture" is as defined above herein. The term "chiral alcohol" refers to an enantiomer of an optically active alcohol, wherein the term "enantiomer" is as defined above herein. The term "alcohol" refers to an organic compound comprising a hydroxyl group attached to a carbon atom. Preferred chiral alcohols for use in the methods described above are menthol and bornol.

Chiral alcohols are cleaved by acid hydrolysis from isolated compounds of Formula (V). Suitable acids for use in this step include hydrochloric or sulfuric acid, preferably 2 molar hydrochloric acid or 1 molar sulfuric acid.

In an alternative aspect, the compound of formula III is

(a) providing a racemic mixture of a compound of Formula III and a compound of Formula VIII

<Formula VIII>

Wherein PG ⁵ is a hydroxyl protecting group and is the same as PG ¹ as defined for Formula III;

(b) separating the compound of formula III from the compound of formula VIII by reacting the mixture as defined in step (a) with an optically active amine

It may be provided using a method comprising a.

A racemic mixture of the compound of Formula III and the compound of Formula VIII can be obtained according to the method described in Scheme 2, wherein the preferred racemic mixture is compound p as described above.

Suitable optically active amines for use in the methods described above include S-alpha-methylbenzylamine, R-(+)-N- (1-naphthylmethyl) -alpha-benzylamine, N- (2-hydroxy) ethyl -Alpha-methyl benzyl amine and 1 (P-tolyl) ethylamine. Other optically active amines suitable for use in the process are commercially readily available from, for example, Aldrich chemical company.

(B) separating the compound of formula III from the compound of formula IV by reacting the mixture of step (a) with the optically active amine initially produces two diastereomeric salts. Such diastereomeric salts are separated by crystallization from a suitable solvent such as acetone or ethyl acetate. The separated salt is treated with an inorganic acid such as 2N hydrochloric acid or 1M sulfuric acid to regenerate the compound of formula III isolated from the enantiomer of formula VIII. The compound of formula III was then recovered by extraction into ethyl acetate, separated from the aqueous layer and concentrated in vacuo to give the enantiomer of formula III.

In a further alternative, the compound of formula III is

(a) providing a racemic mixture of a compound of formula (IX) with a compound of formula (X)

<Formula IX>

<Formula X>

Wherein PG ⁶ and PG ⁷ are the same and each is a hydroxyl protecting group;

(b) reacting the mixture as defined in step (a) with a stereoselective enzyme that affects ester hydrolysis of the compound of formula IX to obtain a compound of formula III

It can be obtained using a method comprising a.

The racemic mixture of the compound of formula (IX) and the compound of formula (X) can be obtained according to the method described in Scheme 2, wherein the preferred racemic mixture is compound o as described above.

Suitable stereoselective enzymes for use in the methods described above are Candida antarctica. antarctica ) lipase B, pig liver esterase, pig pancreatic lipase, or other known stereoselective enzymes that function in a similar manner.

Preparation of PET Tracer

In a further aspect, the present invention provides a method of making a PET tracer of the present invention comprising reacting a precursor compound of Formula I with a suitable source of ¹⁸ F. ^The reaction with ¹⁸ F may be accomplished by nucleophilic substitution of the leaving group present at the R ¹ position of the precursor compound of formula (I). The precursor compound can be labeled in one step by reaction with a suitable source of [ ¹⁸ F] -fluoride ions ( ¹⁸ F ⁻ ), which is generally employed as an aqueous solution from nuclear reaction ¹⁸ 0 (p, n) ¹⁸ F It is obtained and becomes reactive by the addition of cationic counterions and subsequent water removal. Suitable cationic counterions should have sufficient solubility in anhydrous reaction solvent to maintain a solubility of ¹⁸ F. Thus, the counterions used include large but soft metal ions such as rubidium or cesium, kryptand, such as potassium or tetraalkylammonium salts complexed with Kryptofix ™. Preferred counterions are potassium complexed with kryptands such as Cryptofix ™ because of their good solubility in anhydrous solvents and improved ¹⁸ F ⁻ reactivity. ¹⁸ F can also be introduced by O-alkylation of the hydroxyl group at the R ¹ position of the precursor compound with ¹⁸ F (CH ₂ ) ₃ -LG, where LG represents a leaving group as defined above.

A more detailed discussion of the well-known ¹⁸ F labeling technique can be found in Chapter 6 of the "Handbook of Radiopharmaceuticals"(2003; John Wiley and Sons: MJ Welch and CS Redvanly, Eds.).

In a preferred embodiment, the method for producing the PET tracer of the present invention is automated. The [ ¹⁸ F] -radiotracer can be conveniently manufactured in an automated manner by an automated radiosynthesis device. There are several commercially available examples of such devices, including Tracerlab ™ and Fastlab ™ (both available from GE Healthcare Ltd.). Such devices usually include disposable "cassettes", in which radiochemistry is performed, which are mounted to the device to perform radiosynthesis. The cassette usually includes a fluid path, a reaction vessel, and a port for accepting the reagent vial, as well as any solid-phase extraction cartridge used in the cleaning step after radiosynthesis.

Thus, in another aspect the invention

i) a container containing a precursor compound of formula (I) as defined herein; And

ii) means for eluting the vessel of step (i) with a suitable source of ¹⁸ F as defined herein

It provides a cassette for automated synthesis of a PET tracer, as defined herein.

In the cassette of the present invention, suitable and preferred embodiments of the precursor compound of formula (I) and a suitable source of ¹⁸ F are as previously defined herein.

The cassette is

iii) ion-exchange cartridge for removal of excess ¹⁸ F

It may further include.

Radiopharmaceutical composition

In a further aspect, the present invention provides a radiopharmaceutical composition comprising a PET tracer as defined herein together with a biocompatible carrier suitable for mammalian administration.

A "biocompatible carrier" is a fluid, in particular a liquid, in which the PET tracer of the present invention is suspended or dissolved such that the radiopharmaceutical composition is physiologically acceptable, i.e., can be administered to the mammalian body without toxic or excessive discomfort. The biocompatible carrier suitably includes injectable carrier liquids such as sterile, pyrogen-free water for injection; Aqueous solutions such as saline (which may advantageously be balanced such that the final product for injection is isotonic or not hypotonic); One or more tonicity-modulating substances (eg salts of plasma cations with biocompatible counterions), sugars (eg glucose or sucrose), sugar alcohols (eg sorbitol or mannitol), glycols ( For example, glycerol), or other nonionic polyol materials (eg, polyethylene glycol, propylene glycol, etc.). Biocompatible carriers can also include biocompatible organic solvents such as ethanol. Such organic solvents are useful for solubilizing more lipophilic compounds or agents. Preferably, the biocompatible carrier is pyrogen-free water for injection, isotonic saline or aqueous ethanol solution. The pH of the biocompatible carrier for intravenous injection suitably ranges from 4.0 to 10.5.

The radiopharmaceutical composition may be administered parenterally, ie by injection, most preferably in aqueous solution. Such compositions optionally comprise additional ingredients such as buffers; Pharmaceutically acceptable solubilizers (e.g. cyclodextrins or surfactants such as Pluronic, Tween or phospholipids; pharmaceutically acceptable stabilizers or antioxidants (e.g. ethanol, ascorbic acid, gen) Tris acid or para-aminobenzoic acid) When the PET tracer of the present invention is provided as a radiopharmaceutical composition, the method for producing the PET tracer is a step necessary for obtaining a radiopharmaceutical composition, for example, an organic. It may further comprise the removal of the solvent, the addition of biocompatible buffers and optional additional ingredients For parenteral administration, it is also necessary to carry out steps to ensure that the radiopharmaceutical composition is sterile and nonpyrogenic. Such steps are known to those skilled in the art.

PET imaging method

PET tracers of the invention are useful for in vivo detection of PBR receptor expression in a subject. Thus in another aspect, the invention

i) administering a PET tracer to a subject as defined herein;

ii) allowing the PET tracer to bind to PBR in the subject;

iii) detecting a signal emitted by ¹⁸ F included in the combined PET tracer;

iv) generating an image representing the location and / or amount of the signal; And

v) measuring the distribution and extent of PBR expression directly correlated with the signal in the subject

It provides a PET imaging method for measuring the distribution and / or degree of PBR expression in the subject.

The step of "administering" the PET tracer is preferably carried out parenterally, and most preferably intravenously. The intravenous route represents the most efficient way of delivering a PET tracer, which comes into contact with PBR expressed in the subject's central nervous system (CNS) through the body of the subject and thus also across the blood-brain barrier (BBB). Intravenous administration does not exhibit substantial physical disturbances or substantial health risks to the subject. The PET tracer of the present invention is preferably administered as the radiopharmaceutical composition of the present invention as defined herein. No administering step is required for the complete definition of the PET imaging method of the present invention. As such, the PET imaging method of the present invention may also be understood to include steps (ii) to (v) as defined above, wherein the subject of step (ii) is a subject to which the PET tracer of the present invention has been pre-administered Is one of.

After the dosing step and before the detecting step, the PET tracer is allowed to bind to PBR. For example, if the subject is an intact mammal, the PET tracer will move dynamically through the mammal's body to contact various tissues in the body. Once the PET tracer contacts PBR, specific interactions occur such that clearance of PET tracer from tissue with PBR takes longer than clearance from less or no PBR tissue. As a result of the ratio between PET tracers bound to tissue with PBR to PET tracers bound to tissue with or without PBR, a certain time point will be reached that enables detection of PET tracers specifically bound to PBR.

The “detecting” step of the method of the invention involves the detection of the signal emitted by the ¹⁸ F included in the PET tracer by the detector sensing the signal, ie the PET camera. The detecting step can also be understood as acquisition of signal data.

The "generating" step of the method of the invention is performed by a computer applying a reconstruction algorithm to the obtained signal data to produce a dataset. The dataset is then manipulated to produce an image showing the location and / or amount of the signal emitted by ¹⁸ F. The emitted signal is directly related to the expression of PBR so that the "measurement" step can be performed by evaluating the generated image.

A "subject" of the invention can be any human or animal subject. Preferably the subject of the invention is a mammal. Most preferably, the subject is intact mammal body in vivo. In a particularly preferred embodiment, the subject of the invention is a human. In vivo imaging methods can be used to study PBR in healthy subjects or in subjects who know or suspect that they have a pathological condition associated with abnormal expression of PBR (hereinafter “PBR state”). Preferably, the method relates to in vivo imaging of a subject who knows or suspects of having a PBR state, and is therefore useful for methods of diagnosing the condition.

Examples of such PBR conditions that would be useful in vivo imaging include multiple sclerosis, Rasmussen encephalitis, cerebral vasculitis, herpes encephalitis, AIDS-associated dementia, Parkinson's disease, cortical basal degeneration, progressive nuclear palsy, multisystem atrophy, Huntington's disease, muscular dystrophy Sclerosis, Alzheimer's disease, ischemic stroke, peripheral nerve damage, epilepsy, traumatic brain injury, acute stress, chronic stress, neuropathic pain, lung inflammation, chronic obstructive pulmonary disease, asthma, inflammatory bowel disease, rheumatoid arthritis, primary fibromyalgia, Nerve damage, atherosclerosis, kidney inflammation, ischemia-reperfusion injury and cancer, especially colon cancer, prostate cancer or breast cancer. The PET tracer of the present invention is particularly suitable for in vivo imaging of the CNS due to its excellent brain absorption rate.

In alternative embodiments, the PET imaging method of the present invention may be performed repeatedly during the course of a therapeutic regimen for the subject, wherein the regimen comprises administration of a drug to combat PBR status. For example, the PET imaging method of the present invention can be performed before, during and after treatment with drugs that combat PBR conditions. In this manner, the effect of the treatment can be monitored over time. Since PET has excellent sensitivity and resolution, PET is particularly suitable for this application because relatively small changes in the lesion can be observed over time (specific advantages for therapeutic monitoring).

In a further aspect, the present invention comprises a PET imaging method as defined above, with the additional step (vi) of determining the distribution and extent of PBR expression as a result of a particular clinical picture, wherein the PBR is upregulated. Provide diagnostic methods.

In another aspect, the present invention provides a PET tracer as defined herein for use in the above defined PET imaging method and the above defined diagnostic method. The present invention also provides a PET tracer as defined herein for use in the manufacture of a radiopharmaceutical composition as defined herein for use in the above defined PET imaging method and the diagnostic method defined above.

Suitable and preferred aspects of any feature present in the various aspects of the invention are as defined for the feature in the first aspect described herein. The present invention is now illustrated by a series of non-limiting examples.

Brief Description of the Embodiments

Example 1 describes the synthesis of a racemic mixture comprising a precursor compound of formula (I) and an enantiomer of formula (II).

Example 2 describes the synthesis of non-radioactive racemic mixtures comprising non-radioactive analogues of the PET tracer of the present invention together with their alternative enantiomers.

Example 3 describes the synthesis of precursor compound 1 / active enantiomers.

Example 4 describes the synthesis of imaging agent 1 / active enantiomers.

Example 5 describes the synthesis of non-radioactive imaging agent 1 / active enantiomers.

Example 6 describes the method used to determine absolute stereochemistry.

Example 7 describes the in vitro assays used to assess the binding of nonradioactive racemate 1 and its two enantiomers.

Example 8 describes the method used to study the in vitro chiral stability of the PET tracer of the present invention.

Example 9 describes the PET tracer of the invention, alternative enantiomers thereof, and the method used to evaluate the in vivo biodistribution of both racemic mixtures.

Example 10 describes an experiment for evaluating the metabolism of the PET tracer of the present invention and the metabolism of the racemic mixture comprising the PET tracer and its alternative enantiomers.

Example 11 describes the PET tracer of the invention and the in vivo blocking assay used to evaluate a racemic mixture comprising the PET tracer and its alternative enantiomers.

Example 12 describes an animal inflammation model used to evaluate a PET tracer of the invention and a racemic mixture comprising the PET tracer and its alternative enantiomers.

<Brief Description of Drawings>

1 and 4 relate to Example 4 and show radioactive (top) and UV (bottom) HPLC traces obtained using semi-purifying methods for the PET tracer and alternative enantiomers thereof of the present invention, respectively.

2 and 5 relate to Example 4 and show HPLC traces obtained using analytical achiral methods for the PET tracer of the invention and alternative enantiomers thereof, respectively.

3 and 6 relate to Example 4 and show HPLC traces obtained using chiral HPLC methods for the PET tracer of the invention and alternative enantiomers thereof, respectively.

FIG. 7 shows an overlay chromatogram of PET tracer and alternative enantiomers in connection with Example 8, dissolved in acetonitrile at a concentration of 0.1 mg / mL.

8A relates to Example 8 and shows a chromatogram of a PET tracer dissolved in acetonitrile at a concentration of 0.1 mg / mL.

8B shows a chromatogram of PET tracer (0.1 mg / mL) associated with Example 8 and added to human plasma and incubated after extraction.

FIG. 8C shows a chromatogram of PET tracer (0.1 mg / mL) incubated with human plasma and extracted with human plasma.

<List of Abbreviations Used in Examples>

AUFS Absorption Unit Actual Scale

aq aqueous

DCM dichloromethane

DFT density function theory

DMAP 4-dimethylaminopyridine

DMF Dimethylformamide

EDC 1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride

Termination of EOS Synthesis

EtOAc ethyl acetate

FNA facial nerve axon cutting

IPA isopropyl alcohol

IR infrared

LC-MS Liquid Chromatography-Mass Spectrometry

MeCN acetonitrile

MeOH Methanol

NMR nuclear magnetic resonance

OBn benzyloxy

OMs mesylate

OTs Tosylate

PET Positron Emission Tomography

QMA Quaternary Methyl Ammonium

RT room temperature

SFC Supercritical Fluid Chromatography

SPE Solid Phase Extraction

TLC thin layer chromatography

Tol toluene

VCD Vibration Circular Polarization Dichroism

<Examples>

Example 1 Synthesis of Racemic Mixture of Mesylate

Precursor compounds of the present invention (“precursor compound 1”) and alternative enantiomers thereof

Example 1 (a): benzyloxy acetyl chloride (1)

To benzyloxyacetic acid (10.0 g, 60.0 mmol, 8.6 mL) in dichloromethane (50 mL) add oxalyl chloride (9.1 g, 72.0 mmol, 6.0 mL) and DMF (30.0 mg, 0.4 mmol, 32.0 μl), Stir at room temperature for 3 hours. As the reaction proceeded, there was a sudden gas generation, but the gas generation was stopped when the reaction was completed. The dichloromethane solution was concentrated in vacuo to give a gum. The gum was treated with additional oxalyl chloride (4.5 g, 35.7 mmol, 3.0 mL), dichloromethane (50 mL) and 1 drop of DMF. There was a rapid gas evolution and the reaction was stirred for an additional 2 hours. The reaction was then concentrated in vacuo to give 11.0 g (quantitative) of benzyloxy acetyl chloride (1) as a gum. The structure was confirmed by ¹³ C NMR (75 MHz, CDCl ₃ ):

Example 1 (b) 2-benzyloxy-N- (2-chloro-5-methoxy-phenyl) acetamide (2)

Benzyloxy acetyl chloride (1) (11.0 g, 60.0 mmol) and 2-chloro-5-methoxyaniline hydrochloride (11.7 g, 60.2 mmol) in dichloromethane (100 mL) are stirred at 0 ° C. and triethylamine (13.0 g 126.0 mmol, 18.0 mL) was added slowly over 15 minutes. The stirred reaction was allowed to warm to room temperature over 18 hours. There was heavy precipitation of large amounts of triethylamine hydrochloride. The dichloromethane solution was washed with 10% aqueous potassium carbonate (50 mL), dried over magnesium sulfate and concentrated in vacuo to give 2-benzyloxy-N- (2-chloro-5-methoxy-phenyl) acetamide (2 ) 18.9 g (quantitative) were obtained as a gum. The structure was confirmed by ¹³ C NMR (75 MHz, CDCl ₃ ):

Example 1 (c): (2-benzyloxy-ethyl)-(2-chloro-5-methoxyphenyl) amine (3)

2-Benzyloxy-N- (2-chloro-5-methoxy-phenyl) acetamide (2) (18.9 g, 62.0 mmol) in THF (100 mL) was stirred and lithium aluminum hydride (4.9 g, 130.0 mmol) ) Was added slowly over 15 minutes. The first addition of lithium aluminum hydride resulted in the rapid generation of hydrogen gas. The reaction was then heated under reflux for 4 hours and left at room temperature over the weekend. The reaction was then quenched by dropwise addition of water (50 mL) to the stirred solution. There was a vigorous hydrogen evolution, which caused the reaction mixture to reflux. The reaction was then concentrated to a slurry under vacuum. Water (200 mL) and ethyl acetate (200 mL) were added and the mixture was vigorously shaken. The reaction was then filtered through celite to remove the precipitated aluminum hydroxide, the ethyl acetate solution was separated, dried over magnesium sulfate and concentrated in vacuo (2-benzyloxy-ethyl)-(2-chloro-5 18.4 g (quantitative) of -methoxyphenyl) amine (3) was obtained as a gum. The structure was confirmed by ¹³ C NMR (75 MHz, CDCl ₃ ):

Example 1 (d): 3-bromo-2-hydroxy-cyclohex-1-enecarboxylic acid ethyl ester (4)

Ethyl 2-oxocyclohexanecarboxylate (30 g, 176 mmol, 28 mL) was dissolved in diethyl ether (30 mL) and cooled to 0 ° C. under nitrogen. Bromine (28 g, 176 mmol, 9.0 mL) was added dropwise over 15 minutes and the reaction mixture was allowed to warm to room temperature over 90 minutes. The mixture was poured slowly into ice cold saturated aqueous potassium carbonate (250 mL) and extracted with ethyl acetate (3 x 200 mL). The combined organic layers were dried over magnesium sulfate, filtered, concentrated in vacuo and dried for 18 hours on a vacuum line to give 41.4 g of 3-bromo-2-hydroxy-1-enecarboxylic acid ethyl ester (4) ( 94%) was obtained as a yellow oil. The structure was confirmed by ¹³ C NMR (75 MHz, CDCl ₃ ):

Example 1 (e): 3 [(2-benzyloxy-ethyl)-(2-chloro-5-methoxy-phenyl) -amino] -2-hydroxy-cyclohex-1-enecarboxylic acid ethyl ester (5)

(2-benzyloxy-ethyl)-(2-chloro-5-methoxyphenyl) amine (3) (10.0 g, 34.2 mmol) was stirred in dry THF (100 mL) at −40 ° C. under nitrogen and potassium bis (Trimethylsilyl) amide (143.0 mL of 0.5 M solution in toluene, 72.0 mmol) was added over 30 minutes. Then 3-bromo-2-hydroxycyclohex-1-enecarboxylic acid ethyl ester (4) (8.5 g, 34.2 mmol) in dry THF (10 mL) was added and warmed to room temperature over 1.5 hours. It was. Acetic acid (10.0 g, 166 mmol, 10.0 mL) was added and concentrated in vacuo to remove THF. Ethyl acetate (200 mL) and 10% aqueous potassium carbonate (100 mL) were added and the mixture was vigorously shaken. The ethyl acetate solution was separated, dried over magnesium sulfate and concentrated in vacuo to give 3 [(2-benzyloxy-ethyl)-(2-chloro-5-methoxy-phenyl) -amino] -2-hydroxy-cyclo 16.5 g (quant.) Of hex-1-enecarboxylic acid ethyl ester (5) was obtained as a gum, which was used crudely in the next step. HPLC of the crude reaction mixture (Gemini 150 × 4.6 mm, 50 → 95% methanol / water over 20 minutes): 18.9 min (38%), 19.2 min (25%), 23.1 min (28%).

One component of the reaction was identified by ¹³ C NMR (75 MHz, CDCl ₃ ):

Example 1 (f): 9- (2-benzyloxy-ethyl) -8-chloro-5-methoxy-2,3,4,9-tetrahydro-1H-carbazole-4-carboxylic acid ethyl ester (6)

Zinc chloride (7.1 g, 52.0 mmol) was added to 3 [(2-benzyloxy-ethyl)-(2-chloro-5-methoxy-phenyl) -amino] -2- in anhydrous diethyl ether (150 mL) under nitrogen. To hydroxy-cyclohex-1-enecarboxylic acid ethyl ester (5) (8.0 g, 17.0 mmol) and heated at reflux for 5.5 h. As the reaction was refluxed, a dense brown viscous oil formed in the reaction. The reaction was then cooled and the supernatant diethyl ether was decanted, ethyl acetate (100 mL) was added and washed with 2N HCl (50 mL) and 10% aqueous potassium carbonate (50 mL). The diethyl ether layer was separated, dried over magnesium sulfate and concentrated in vacuo to give an oil (2.0 g). The crude material was purified by silica gel chromatography eluting with gasoline (A): ethyl acetate (B) (10 → 40% (B), 340 g, 22 CV, 150 mL / min) to give 9- (2-benzyl). 1.8 g of oxy-ethyl) -8-chloro-5-methoxy-2,3,4,9-tetrahydro-1H-carbazole-4-carboxylic acid ethyl ester (6) were obtained. The thick viscous brown layer was treated with ethyl acetate (100 mL) and 2N HCl (50 mL). The ethyl acetate solution was separated, washed with 10% aqueous potassium carbonate (50 mL), dried over magnesium sulfate and concentrated in vacuo to give an oil (5.2 g). Diethyl ether (100 mL) and anhydrous zinc chloride (7.0 g) were added. The mixture was heated at reflux for an additional 5 days. The ether layer was decanted from dark gum, washed with 2N HCl (50 mL), dried over magnesium sulfate and concentrated in vacuo to give a gum (2.8 g). The gum was purified by silica gel chromatography eluting with gasoline (A): ethyl acetate (B) (5 → 35% (B), 340 g, 150 mL / min) to give 9- (2-benzyloxy-ethyl) 2.1 g of -8-chloro-5-methoxy-2,3,4,9-tetrahydro-1H-carbazole-4-carboxylic acid ethyl ester (6) was obtained. The total material obtained was 9- (2-benzyloxy-ethyl) -8-chloro-5-methoxy-2,3,4,9-tetrahydro-1H-carbazole-4-carboxylic acid ethyl ester (6 ) 4.1 g (50%). The structure was confirmed by ¹³ C NMR (75 MHz, CDCl ₃ ):

Example 1 (g): 9- (2-benzyloxy-ethyl) -8-chloro-5-methoxy-2,3,4,9-tetrahydro-1H-carbazole-4-carboxylic acid (7 )

9- (2-benzyloxy-ethyl) -8-chloro-5-methoxy-2,3,4,9-tetrahydro-1H-carbazole-4-carboxylic acid ethyl ester in ethanol (50 mL) 6) To (2.0 g, 4.1 mmol) was added sodium hydroxide (1.1 g, 27.1 mmol) and water (5 mL) and heated at 80 ° C. for 18 h. The ethanol was then removed by evaporation in vacuo and the residue was partitioned between diethyl ether (50 mL) and water (50 mL). The diethyl ether layer was separated, dried over magnesium sulfate and concentrated in vacuo to give a gum (71.0 mg). The aqueous layer was acidified to pH 1 with 2 N HCl (20 mL) and extracted with dichloromethane (2 × 100 mL). The dichloromethane layer was dried over magnesium sulfate and concentrated in vacuo to give 9- (2-benzyloxy-ethyl) -8-chloro-5-methoxy-2,3,4,9-tetrahydro-1H-carbazole- 1.6 g (87%) of 4-carboxylic acid (7) was obtained as a foam. The structure was confirmed by ¹³ C NMR (75 MHz; CDCl ₃ ):

Example 1 (h): 9- (2-benzyloxy-ethyl) -8-chloro-5-methoxy-2,3,4,9-tetrahydro-1H-carbazole-4-carbonyl chloride (8 )

9- (2-benzyloxy-ethyl) -8-chloro-5-methoxy-2,3,4,9-tetrahydro-1H-carbazole-4-carboxylic acid (7) (1.5 g, 3.7 mmol ) Was dissolved in dichloromethane (50 mL), oxalyl chloride (700 mg, 5.5 mmol, 470 μl) and DMF (1 drop) were added and the reaction stirred at 20 ° C. for 2 hours. There was a moderate gas evolution for about 30 minutes as the reaction progressed. The reaction was then concentrated in vacuo to give 9- (2-benzyloxy-ethyl) -8-chloro-5-methoxy-2,3,4,9-tetrahydro-1H-carbazole-4-carbonyl chloride ( 8) was obtained as a gum, which was used without purification in the next step. The structure was confirmed by ¹³ C NMR (75 MHz; CDCl ₃ ):

Example 1 (i): 9- (2-benzyloxy-ethyl) -8-chloro-5-methoxy-2,3,4,9-tetrahydro-1H-carbazole-4-carboxylic acid diethyl Amide (9)

Then 9- (2-benzyloxy-ethyl) -8-chloro-5-methoxy-2,3,4,9-tetrahydro-1H-carbazole-4-carbonyl chloride (8) (1.6 g, 3.7 mmol) was dissolved in dichloromethane (50 mL), cooled to 0 ° C., stirred and diethylamine (810 mg, 11.0 mmol, 1.1 mL) was added dropwise. The reaction was allowed to warm to room temperature over 18 hours. The reaction mixture was then washed with 10% aqueous potassium carbonate (50 mL), separated, dried over magnesium sulphate and concentrated in gum under vacuum. The crude material was crystallized from diethyl ether to give 9- (2-benzyloxy-ethyl) -8-chloro-5-methoxy-2,3,4,9-tetrahydro-1H-carbazole-4-carboxylic acid 1.2 g (71%) of diethylamide (9) were obtained as a white crystalline solid. The structure was confirmed by ¹³ C NMR (75 MHz; CDCl ₃ ):

Example 1 (j): 9- (2-benzyloxy-ethyl) -5-methoxy-2,3,4,9-tetrahydro-1H-carbazole-4-carboxylic acid diethylamine (10)

9- (2-benzyloxy-ethyl) -8-chloro-5-methoxy-2,3,4,9-tetrahydro-1H-carbazole-4-carboxylic acid diethylamide in methanol (100 mL) (9) (1.0 g, 2.1 mmol) was shaken with 10% palladium (1.0 g) on charcoal, triethylamine (2.9 mg, 2.9 mmol, 4 μl) at 55 ° C. under hydrogen gas atmosphere for 18 hours. The reaction was then filtered through a pad of celite and the filtrate was concentrated in vacuo to afford a gum (908 mg). The gum was then dissolved in dichloromethane (100 mL) and washed with 5% aqueous potassium carbonate solution (50 mL). The dichloromethane solution was then separated, dried over magnesium sulfate and concentrated in vacuo to afford a gum. The gum was then crystallized from diethyl ether (50 mL) and the crystals collected by filtration to give 9- (2-benzyloxy-ethyl) -5-methoxy-2,3,4,9-tetrahydro-1H- 523 mg (57%) of carbazole-4-carboxylic acid diethylamine (10) were obtained. The structure was confirmed by ¹³ C NMR (75 MHz; CDCl ₃ ):

 Example 1 (k): 9- (2-hydroxyethyl) -5-methoxy-2,3,4,9-tetrahydro-1H-carbazole-4-carboxylic acid diethylamine (11)

9- (2-benzyloxy-ethyl) -5-methoxy-2,3,4,9-tetrahydro-1H-carbazole-4-carboxylic acid diethylamine (10) in methanol (50 mL) ( 1.0 g, 2.1 mmol) was shaken for 18 h at 55 ° C. with 10% palladium (300 mg) on charcoal and excess hydrogen gas. The reaction was then filtered through a pad of celite and the filtrate was concentrated in vacuo to give 9- (2-hydroxyethyl) -5-methoxy-2,3,4,9-tetrahydro-1H-carbazole 578 mg (100%) of 4-carboxylic acid diethylamine (11) were obtained as a foam. The structure was confirmed by ¹³ C NMR (75 MHz; CDCl ₃ ):

Example 1 (l): Methanesulfonic acid 2- (4-diethylcarbamyl-5-methoxy-1,2,3,4-tetrahydro-carbazol-9-yl) ethyl ester

9- (2-hydroxyethyl) -5-methoxy-2,3,4,9-tetrahydro-1H-carbazole-4-carboxylic acid diethylamine (11) in dichloromethane (30 mL) ( 478 mg, 1.4 mmol) was cooled to 0 ° C., methanesulfonyl chloride (477 mg, 4.2 mmol, 324 μl) and triethylamine (420 mg, 4.2 mmol, 578 μl) were added and warmed to room temperature overnight. . The reaction was washed with 5% aqueous potassium carbonate solution. The layers were separated. The combined organic layers were dried over magnesium sulphate and concentrated in vacuo to give a gum (696 mg). Crude was purified by silica gel chromatography eluting with gasoline (A): ethyl acetate (B) (75 → 100% B, 22 CV, 120 g, 85 mL / min) to methanesulfonic acid 2- (4-di Ethylcarbamyl-5-methoxy-1,2,3,4-tetrahydro-carbazol-9-yl) ethyl ester was obtained as a gum, which was crystallized from diethyl ether to give 346 mg (59%) of a colorless solid. Obtained. The structure was confirmed by ¹³ C NMR (75 MHz; CDCl ₃ ):

Example 2: Synthesis of racemic mixtures of non-radioactive PET tracers of the present invention and their alternative enantiomers

Example 2 (a): fluoroethyl tosylate (12)

2-fluoroethanol (640 mg, 10 mmol, 0.6 mL) was dissolved in pyridine (10 mL) under nitrogen. The solution was stirred at 0 ° C. and tosyl chloride (4.2 g, 21.8 mmol) was added portionwise to the solution over 30 minutes while maintaining the temperature below 5 ° C. The reaction was stirred at 0 ° C for 3 h. Ice was added slowly followed by water (20 mL). The reaction mixture was extracted with ethyl acetate and washed with water. Excess pyridine was removed by washing with 1N HCl solution until the aqueous layer became acidic. Excess tosyl chloride was removed by washing with 1 M aqueous sodium carbonate. The organic layer was washed with brine, dried over magnesium sulfate and concentrated in vacuo to give 2.1 g (98%) of fluoroethyl tosylate (12) as a colorless oil. The structure was confirmed by ¹³ C NMR (75 MHz, CDCl ₃ ):

Example 2 (b): 2-chloro-5-methoxy-phenyl) (2-fluoroethyl) amine (13)

2-chloro-5-methoxyaniline hydrochloride (5.0 g, 26.0 mmol) was dissolved in DMF (50 mL) and sodium hydride (2.3 g, 60% in oil, 57.0 mmol) was added. The reaction was stirred at rt for 30 min under nitrogen. Fluoroethyl tosylate (12) (6.7 g, 31.0 mmol) in DMF (5 mL) was added dropwise and the reaction was stirred at rt for 2 h. The reaction was then heated at 100 ° C. for 18 hours. The reaction was cooled and the solvent removed under reduced pressure. The residue was dissolved in ethyl acetate (100 mL) and washed with water (2 x 100 mL). The organics were collected, dried over magnesium sulphate and concentrated in vacuo to give a brown oil which was purified by silica gel chromatography in gasoline (A): ethyl acetate (B) (5 → 30% (B), 330 g, Purification by eluting with 18.1 CV, 120 mL / min) gave 1.3 g (25%) of 2-chloro-5-methoxy-phenyl) (2-fluoroethyl) amine (13) as a yellow oil. The structure was confirmed by ¹³ C NMR (75 MHz; CDCl ₃ ):

Example 2 (c): 3-[(2-chloro-5-methoxy-phenyl)-(2-fluoroethyl) amino] -2-hydroxy-cyclohexyl-1-enecarboxylic acid ethyl ester ( 14)

A solution of 2-chloro-5-methoxy-phenyl) (2-fluoroethyl) amine (13) (6.1 g, 30.0 mmol) in THF (170 mL) was cooled to -40 ° C. Potassium bis (trimethylsilyl) amide (126.0 mL of a 0.5 M solution in toluene, 63.0 mmol) was added dropwise and the reaction stirred at -40 ° C for 30 minutes. 3-bromo-2-hydroxy-cyclohex-1-enecarboxylic acid ethyl ester (4; prepared according to example 1 (d)) (7.4 g, 30.0 mmol) in THF (30 mL) -40 It was added dropwise at ℃. The cold bath was removed and the reaction stirred at room temperature for 4 hours. The reaction was quenched with brine (300 mL), extracted with ethyl acetate (2 × 400 mL), dried over magnesium sulfate and concentrated in vacuo to afford 3-[(2-chloro-5-methoxy-phenyl)-( 12.0 g (quant.) Of 2-fluoroethyl) amino] -2-hydroxy-cyclohexyl-1-enecarboxylic acid ethyl ester (14) was obtained as a brown oil, which was used crudely in the next step. The structure as a mixture of isomers was confirmed by ¹ H NMR (300 MHz, CDCl ₃ ):

Example 2 (d) 8-chloro-9- (2-fluoroethyl) -5-methoxy-2,3,4,9-tetrahydro-1H-carbazole-4-carboxylic acid ethyl ester (15 )

Synthesis of 8-chloro-9- (2-fluoro-ethyl) -5-methoxy-2,3,4,9-tetrahydro-1H-carbazole-4-carboxylic acid ethyl ester (15) first An attempt was made using the conditions described in WO 2003/014082. A solution of 2-chloro-5-methoxy-phenyl) (2-fluoroethyl) amine (13; prepared according to example 2 (b)) (600 mg, 3.8 mmol) in dry THF (20 mL) was iced It was cooled in and treated with potassium bis (trimethyl silyl) amide (16 mL of 0.5 M solution in toluene, 8.0 mmol). After 30 minutes, 3-bromo-2-hydroxy-cyclohex-1-enecarboxylic acid ethyl ester in THF (4 mL) (4; prepared according to example 1 (d)) (1.04 g, 4.2 mmol ) Was added and the reaction was allowed to warm to room temperature over 2 hours. The reaction was quenched with saturated ammonium chloride solution and extracted twice with ether. The extract was washed with water, brine, dried and concentrated in vacuo. The crude material was purified by silica gel chromatography eluting with gasoline (A) and ethyl acetate (B) (2.5 → 50% B, 50 g, 25 CV, 40 mL / min). The main spot was a mixture of three compounds. The mixture was refluxed overnight with dry zinc chloride (1.7 g, 12.6 mmol) in toluene (20 mL). The reaction was concentrated in vacuo and the residue was partitioned between 1 N HCl (25 mL) and ethyl acetate (25 mL) and then extracted once more with ethyl acetate. The organic layer was washed with water and brine, dried and concentrated in vacuo to give a brown oil. ¹ H NMR indicated that this was a mixture of several compounds. TLC on silica did not separate the mixture into separate spots within the range of solvent. In comparison of ¹ H NMR of mixtures containing certified samples, the mixture yielded an estimated 25% of 8-chloro-9- (2-fluoro-ethyl) -5-methoxy-2,3,4,9-tetrahydro -1H-carbazole-4-carboxylic acid ethyl ester (15).

Subsequently, a modification method was performed. 3-[(2-Chloro-5-methoxy-phenyl)-(2-fluoroethyl) amino] -2-hydroxy-cyclohexyl-1-enecarboxylic acid ethyl ester (14) (12.2 g, 30.0 mmol) was dissolved in diethyl ether (250 mL) and zinc chloride (16.4 g, 120.0 mmol) was added. The reaction was heated at reflux for 16 h. Ethyl acetate (500 mL) was added to dissolve all materials, washed with 2N HCl (200 mL), water (200 mL), 10% aqueous potassium carbonate (200 mL), dried over magnesium sulfate and dried in vacuo. Concentrated. Crude was purified by silica gel chromatography eluting with gasoline (A): ethyl acetate (B) (5 → 20% B, 12 CV, 10 g, 100 mL / min) to obtain 8-chloro-9- (2 -Fluoroethyl) -5-methoxy-2,3,4,9-tetrahydro-1H-carbazole-4-carboxylic acid ethyl ester (15) 5.3 g (50% over 2 steps) as a yellow solid Obtained as. The structure was confirmed by ¹³ C NMR (75 MHz, CDCl ₃ ):

Example 2 (e): 9- (2-fluoroethyl) -5-methoxy-2,3,4,9-tetrahydro-1H-carbazole-4-carboxylic acid ethyl ester (16)

8-Chloro-9- (2-fluoroethyl) -5-methoxy-2,3,4,9-tetrahydro-1H-carbazole-4-carboxylic acid ethyl ester (15) (5.3 g, 15.0 mmol) was dissolved in methanol (180 mL) and triethylamine (1.8 g, 18.0 mmol, 2.5 mL) and 10% Pd / C (2 g in methanol (20 mL)) were added. The mixture was placed in a Parr hydrogenator and shaken under hydrogen atmosphere for 18 hours. The reaction was filtered through a pad of celite, washed with methanol and the solvent removed in vacuo. The residue is dissolved in ethyl acetate (300 mL), washed with 10% aqueous potassium carbonate (200 mL), dried over magnesium sulphate and concentrated in vacuo to give 9- (2-fluoroethyl) -5-methoxy 4.2 g (88%) of -2,3,4,9-tetrahydro-1H-carbazole-4-carboxylic acid ethyl ester (16) were obtained as a light brown solid. The structure was confirmed by ¹³ C NMR (75 MHz, CDCl ₃ ):

HPLC (Gemini 150 × 4.6 mm, 50 → 95% methanol / water over 20 minutes) 13.6 minutes (94%).

Example 2 (f): 9- (2-fluoroethyl) -5-methoxy-2,3,4,9-tetrahydro-1H-carbazole-4-carboxylic acid (17)

8-chloro-9- (2-fluoroethyl) -5-methoxy-2,3,4,9-tetrahydro-1H-carbazole-4-carboxylic acid ethyl ester (16) (380 mg, 1.2 mmol) was dissolved in ethanol (4 mL). A solution of sodium hydroxide (580 mg, 14.5 mmol) dissolved in 6 mL of water was added. The reaction mixture was heated at reflux overnight. The solvent was removed in vacuo, the crude mixture was diluted with water, acidified with 2N HCl until acidic and washed with dichloromethane. The organics were combined, dried over magnesium sulfate and concentrated in vacuo to give 9- (2-fluoroethyl) -5-methoxy-2,3,4,9-tetrahydro-1H-carbazole-4-carboxylic acid (17) 347 mg (quantitative) were obtained as off-white solid, which was used crude as next step. The structure was confirmed by ¹³ C NMR (75 MHz; CDCl ₃ ):

Example 2 (g): 9- (2-fluoroethyl) -5-methoxy-2,3,4,9-tetrahydro-1H-carbazole-4-carbonyl chloride (18)

9- (2-fluoroethyl) -5-methoxy-2,3,4,9-tetrahydro-1H-carbazole-4-carboxylic acid (17) in dry dichloromethane (2 mL) (347 mg , 1.2 mmol) was stirred under nitrogen. Oxalyl chloride (453 mg, 3.6 mmol, 300 μl) was added followed by 1 drop of DMF. The reaction mixture was stirred at room temperature under nitrogen for 2 hours and then evaporated in vacuo to give 9- (2-fluoroethyl) -5-methoxy-2,3,4,9-tetrahydro-1H-carbazole- 371 mg (quantitative) of 4-carbonyl chloride were obtained as a gum, which was used without purification in the next step. The structure was confirmed by ¹³ C NMR (75 MHz, CDCl ₃ ):

Example 2 (h): 9- (2-Fluoroethyl) -5-methoxy-2,3,4,9-tetrahydro-1H-carbazole-4-carboxylic acid diethyl amide

9- (2-fluoroethyl) -5-methoxy-2,3,4,9-tetrahydro-1H-carbazole-4-carbonyl chloride (18) (371 mg, 1.2 mmol) was converted to dichloromethane ( 2 mL) and cooled to 0 ° C. Diethylamine (177 mg, 2.4 mmol, 250 μl) was then added and the reaction stirred overnight at room temperature. The reaction was quenched with 10% aqueous potassium carbonate (2 mL). The dichloromethane layer was collected via a phase separator and then concentrated in vacuo. The crude material was purified by silica gel chromatography eluting with gasoline (A): ethyl acetate (B) (50 → 100% (B), 50 g, 35.2 CV, 40 mL / min) to give a pale yellow solid. . The solid is then triturated with a minimum amount of diethyl ether to give 9- (2-fluoroethyl) -5-methoxy-2,3,4,9-tetrahydro-1H-carbazole-4-carboxylic acid 240 mg (58%) of diethyl amide were obtained. The structure was confirmed by ¹³ C NMR (75 MHz, CDCl ₃ ):

Example 3: Synthesis of Precursor Compound 1 and Alternative Enantiomers thereof

A racemic mixture of precursor compound 1 and its alternative enantiomers (obtained as described in Example 1) was obtained with Kromasil Amycoat (250 × 10 mm, 5 μm, 100 μs column, using 30% IPA, 40 Separated into its enantiomers using chiral supercritical fluid (CO ₂ ) chromatography on C, 13 mL / min, run time 6 min). 60 mg of racemate was dissolved in 1,4-dioxane (2 mL) and injected at once up to 200 μl for each run. Baseline separation between the two isomers was achieved. Enantiomeric purity of two separate enantiomers on an IC (250 × 4.6 mm, 5 μm, isocratic drive, 80: 20-MeOH: IPA, 0.5 mL / min and room temperature) from Chiral Technologies Measurement by analytical HPLC showed 99.5% enantiomeric purity of each enantiomer.

Example 4 Synthesis of PET Tracer of the Invention and Alternative Enantiomers thereof

Precursor Compound 1 and its enantiomers obtained according to Example 3 were labeled at ¹⁸ F using FastLab ™ (GE Healthcare) cassette.

[ ¹⁸ F] fluoride supplied from GE Healthcare of GE PETrace cyclotron was captured in a QMA cartridge. K222 (8 mg), KHCO ₃ (200 μl, 0.1M aqueous) and MeCN (1 ml) were added to Elution Vial 1. The QMA cartridge was eluted using 0.6 ml of eluent from elution vial 1. ^{The 18} F eluent was dried at 100 ° C. for 20 minutes, then cooled to 86 ° C. and then the precursor was added.

3 mg of each precursor compound was dissolved in 1.6 ml of CH ₃ CN. 1 ml of this solution was added to the reaction vessel. The reaction vessel was heated at 100 ° C. for 15 minutes. The reaction vessel was then washed with 2 ml of water.

Semi-prep HPLC was performed as follows:

Analytical achiral HPLC was performed as follows:

Analytical chiral HPLC was performed as follows:

The EOS yield for the PET tracer of the present invention was 32% and for its enantiomer 19%.

Example 5 Synthesis of Non-Radioactive Analogs and Alternative Enantiomers of the PET Tracer of the Invention

The racemic mixture of the non-radioactive PET tracer of the present invention and its alternative enantiomers (obtained as described in Example 2) was subjected to chromamaric amicoat (250 × 10 mm, 5 μm, 100 μs column, 20% IPA, Separated into its enantiomer using chiral supercritical fluid (CO ₂ ) chromatography (SFC) on 40 ° C., 14 mL / min, run time 6 min). 100 mg of the racemic mixture was dissolved in 1,4-dioxane (2.5 mL) and injected at once up to 200 μl for each run. Fractions were cut over time, confirming that no mixed fractions were collected. Enantiomeric purity of two separate enantiomers on an IC (250x4.6 mm, 5 μm, isocratic driven, 80: 20-MeOH: IPA, 0.5 mL / min and room temperature) from Chiral Technologies was analyzed by analytical HPLC. Upon measurement, 99.5% enantiomeric purity of each enantiomer was shown.

Example 6 Determination of Absolute Stereochemistry by Vibrating Circle Dichroism

Precursor compound 1 and its enantiomers were tested as well as non-radioactive analogs and enantiomers thereof of the PET tracer of the present invention. Each test compound was dissolved in CDCl ₃ (5 mg / 0.12 mL for PET Tracer and its enantiomers; 5 mg / 0.15 mL for Precursor Compound 1 and its enantiomers), with a BaF2 window and a route length of 100 It was placed in a cell of μm. ChiralIRTM VCD Spectrometer with DuaLPE Auxiliary Instrument with IR and VCD spectra at 4 cm ^-1 resolution, 11h acquisition for each sample and optimized at 1400 cm ^-1 (BioTools, Inc.) Recorded on. IR of solvent was collected for 150 scans. Solvent-subtracted IR and enantiomer-subtracted VCD spectra were collected. Optical rotation (OR) of each test compound was measured at 590 nm and 25 ° C. using a Jasco DIP-370 polarimeter.

In each case the (R) -coordination was built with Hyperchem (Hypercube, Inc., Gainesville, FL). Conformation searches were performed with hyperchem for the entire structure at the molecular dynamics level. Geometry, frequency, and IR and VCD intensity calculations are based on DFT levels (B ₃ LYP function / 6-31G (d)) using Gaussian 09 (Gaussian Inc., Willingford, CT). Set). The calculated frequency was adjusted to 0.97 and the IR and VCD intensities were converted to Lorentzian bands with 6 cm ⁻¹ half width for comparison with the experiment.

Regarding the PET tracer and its enantiomers, Gaussian calculation of 36 isoforms revealed 10 isoforms with energy within 1 kcal / mol from the lowest-energy isoform. The optimal geometry of the four isoforms calculated at the lowest energy was estimated in the (R) -coordination and the observed VCD and IR spectra were compared with the spectra of the ten isoforms calculated at the lowest energy. Based on the overall agreement in the observed VCD pattern of the ten lowest energy isoforms and the Boltzmann sum of the estimated spectra, the absolute configuration of the non-radioactive analogues of the PET tracer of the present invention is assigned to (S), Its enantiomer was assigned as (R). These assignments were evaluated with the CompareVOA program (BioTools) and the confidence level of these assignments is 100% based on the current database containing the previous 89 correct assignments for different chiral structures.

Regarding precursor compound 1 and its enantiomers, Gaussian calculation of 36 isoforms revealed nine isoforms with energy within 1 kcal / mol from the lowest-energy isoform. Based on the observed VCD pattern of the nine lowest energy isoforms and the overall agreement in the Boltzmann sum of the estimated spectra, the absolute configuration of precursor compound 1 is assigned to (S)-and its enantiomer is to (R)- Was assigned as. These assignments were evaluated with the CompAVOA program and, based on the current database containing the previous 89 correct assignments for the different chiral structures, the confidence level of these assignments was 96%. This assignment is consistent with the assignment of the arrangement of non-radioactive analogs of the PET tracer of the present invention.

Example 7: In Vitro Efficacy Assays

Affinity to PBR was screened using a method adapted by Le Fur et al. (Life Sci. 1983; USA 33: 449-57). Non-radioactive analogues and associated racemates of the PET tracer of the present invention were tested. Each test compound (50 mM Tris-HCl (pH 7.4), dissolved in 10 mM MgCl ₂ containing 1% DMSO) was 0.3 nM [ ³ H for binding to Wistar rat heart PBR or human PBR. Competition with PK-11195. The reaction was performed at 25 ° C. for 15 minutes in 50 mM Tris-HCl, pH 7.4, 10 mM MgCl ₂ . Each test compound was screened at six different concentrations in the range of 300 times the estimated K _i ambient concentration. The following data were observed.

Example 8: In Vitro Chiral Stability Assay

Non-radioactive PET tracers of the invention obtained according to Example 2 were incubated in human plasma or in rat liver S9 fractions for up to 4 hours (37 ° C.). The enantiomer was extracted from the biological material by precipitating the protein. The solid precipitate was separated from the liquid phase and it was dried by evaporation. The dry residue was dissolved in acetonitrile.

A Dionex Ultimate 3000 HPLC system consisting of two pumps (micro pump LPG-3000 and Ultimate 3000 pump), UV / visible detector, auto sampler and two switching valves was applied to this study. . One switching valve connected the two pumps with the auto sampler. This setting makes it possible to use one of the pumps for injection into the column. The pump used for injection was connected only to the SPE column. After infusion and elution, the SPE column was washed using an infusion pump. The system was prepared for fresh injection while chiral analysis was in progress.

Another switching valve connected the analysis column and the SPE column. After holding the material on the SPE column, the valve was switched and the analytical pump eluted the material from the SPE column into the analytical chiral column. The flow direction of the eluent was opposite the flow direction of the retention liquid. The assay pump was connected only to the assay system and waited for the analyte until the start of elution. The two-step method was optimized by varying both the run time on the SPE column and the elution time from this column.

Analytical column: Chiralpak IC 0.46 × 25 cm with a pre-column of 0.4 × 1 cm of the same material.

SPE column: LiChrospher ADS RP-4 25 × 4 mm (RAM column), 25 μm particles. MW cutoff: 15 kDa (Merck).

Mobile phase: A: 10 mM ammonium acetate (pH 7); B: 1: 1 MeCN: MeOH.

The flow rate was 300 (microliters) / minute.

Detection: UV-detection at 230 nm.

Retention on SPE Columns: When holding the analytes on the SPE column, an isocratic mode using 10% MeCN in 50 mM ammonium acetate was applied. Retention was continued for 4 minutes, then the valve was switched.

Elution and Separation from SPE Column: Elution was started using a mobile phase mixture of 10% MeCN and 90% 10 mM ammonium acetate. After 5 minutes the switch on the valve was returned to the SPE column, which was washed with 90% MeCN / MeOH in buffer. Gradient on the assay column started with 65% organic phase in buffer and changed to 85% organic phase in buffer for 26.5 minutes. The analytical column was washed with 70% MeCN / MeOH for 3 minutes and then stabilized at 10% MeCN / MeOH to prepare the separation system for the next injection. The total run time was 40 minutes.

PET tracers in plasma did not show any chromatographic changes after incubation for 4 hours. The chromatographic results of the non-incubated samples in plasma and the reference solution of the PET tracer of the present invention were compared. No racemization was observed.

The PET tracer of the present invention in the rat liver S9 fraction was not racemized even after incubation for 4 hours.

Example 9: In vivo Biodistribution

The PET tracer of the invention, its alternative enantiomers and racemic mixtures of the two were tested in an in vivo biodistribution model.

Adult male Wistar rats (200-300 g) were injected with 1-3 MBq of test compound via the lateral tail vein. At 2, 10, 30, or 60 minutes after injection (n = 3), rats were euthanized and tissue or body fluids sampled to measure radioactivity on a gamma counter.

The following data were observed:

Example 10 In Vivo Metabolic Studies

The amount of brain or plasma activity due to the parent test compound was tested up to 1 hour after administration. PET tracers of the present invention and their related racemates were test compounds.

Adult male Wistar rats (150-200 g) were injected with approximately 20 MBq of test compound. Brain and plasma samples were analyzed by HPLC at pi 10, 30 and 60 minutes. The following HPLC conditions were used:

The following data records were observed (where “pi” means after injection):

Example 11: In vivo Blocking Assay

In vivo biodistribution of the PET tracer of the present invention was tested prior to pre-administration of each of its non-radioactive analogues or after pre-administration of known PBR-specific ligands (PK11195) compared to its associated racemates. .

Adult male Wistar rats (200-300 g) were injected with approximately 3-4 MBq of test compound via the lateral tail vein. PK11195 or non-radioactive analogs (both 3 mg / kg) were administered 5 minutes before the radiolabeled test compound. Thirty minutes after injection, rats were euthanized and tissue or body fluids sampled to measure radioactivity on a gamma counter.

The following data records were observed:

Example 12 Facial Nerve Axontomy Model of Inflammation

Binding of neuroinflammatory to the focal site was tested by autoradiography. The test compound was the PET tracer of the present invention and its related racemates.

Male Wistar rats (200-300 g) are used as naive or by Graeber and Kreutzberg, J Neurocytol 1986; 15: 363-373, rats that experienced facial nerve axon cutting were used according to the procedure described. Various tissues, including brainstem and olfactory bulbs, were harvested from animals, frozen rapidly in isopentane and then stored at −70 ° C. until application. Tissues were cut (12 μm) and thawed-mounted on Superfrost plus slides. The slides were stored at -70 ° C until use.

After the slides were air dried, the slides were pre-incubated for 5 minutes at room temperature in Tris-HCl buffer (170 mM, pH 7.4). Tris-HCl buffer (170 mM, pH 7.4) containing 8 GBq / ml of test compound after addition of 1000-fold excess non-radioactive PK11195 (1 μM), or non-radioactive PET tracer of the present invention (1 μM) Incubate for 60 minutes with. The sections were then quenched by washing twice in ice cold buffer (Tris-HCl, 170 mM, pH 7.4) for 5 minutes each, then the slides were washed simply by dipping in distilled water. The slides were then dried in air and exposed to x-ray film. When exposed to an x-ray film, the reference sample is included and the reference sample (20 μ) is removed from the solution for in vitro autoradiography, placed on filter paper (tap on glass), and with sections Exposed. The film was exposed for 24 hours and the MCID software was used to display blocked samples, references and backgrounds as well as regions of interest around specific anatomical structures, using density gradient scales such as calibration curves adjusted for reference samples. The data was analyzed by plotting it.

The following data records were observed.

Claims (23)

Positron-emitting tomography (PET) tracers with the following chemical structures.

Precursor compounds of formula (I) for the preparation of PET tracers as defined in claim 1.
(I)

Wherein R ¹ is hydroxyl or a leaving group. The precursor compound of claim 2, wherein the leaving group is selected from mesylate, tosylate and triflate. The precursor compound of claim 3, wherein the leaving group is mesylate. (i) providing a racemic mixture of a precursor compound of formula (I) and a compound of formula (II) as defined in any of claims 2 to 4
<Formula II>

Wherein R ² is as defined for R ¹ in any one of claims 2 to 4 and R ¹ and R ² are the same;
(ii) separating the precursor compound of formula (I) from the compound of formula (II)
A first process for preparing a precursor compound of formula (I) as defined in claim 2, comprising: 6. The method of claim 5, wherein said separating step comprises high performance liquid chromatography, supercritical fluid chromatography, mock layer chromatography. (i) providing a compound of formula III
<Formula III>

Wherein PG ¹ is a hydroxyl protecting group;
(ii) converting the compound of Formula III into its corresponding acid chloride;
(iii) reacting the acid chloride obtained in step (ii) with diethylamide to obtain a compound of formula
<Formula IV>

Wherein PG ² is a hydroxyl protecting group and is the same as PG ¹ ;
(iv) deprotecting the compound of formula IV obtained in step (iii) to obtain a hydroxyl derivative;
(v) optionally adding a leaving group as defined in any one of claims 2 to 4 for formula (I).
Wherein the precursor compound of formula (I) is produced from step (iv) or step (v), wherein the second of the precursor compound of formula (I) as defined in any one of claims 2 to 4 Manufacturing method. The method of claim 7, wherein said step (i) to provide a compound of formula III,
(a) providing a racemic mixture of a compound of formula (V) with a compound of formula (VI)
<Formula V>

&Lt; Formula (VI)

(Wherein
R ¹ is chiral alcohol;
PG ³ and PG ⁴ are the same, each being a hydroxyl protecting group);
(b) separating the compound of formula V from the compound of formula VI;
(c) using acidic conditions to remove R ¹ from the isolated compound of formula V, thereby producing a compound of formula III
Method comprising a. The method of claim 7, wherein said step (i) to provide a compound of formula III,
(a) providing a racemic mixture of a compound of Formula III and a compound of Formula VIII
<Formula VIII>

Wherein PG ⁵ is a hydroxyl protecting group and is the same as PG ¹ as defined in claim 7;
(b) separating the compound of formula III from the compound of formula VIII by reacting the mixture as defined in step (a) with an optically active amine
Method comprising a. The method of claim 9, wherein the optically active amine is S-alpha-methylbenzylamine, R-(+)-N- (1-naphthylmethyl) -alpha-benzylamine, N- (2-hydroxy) ethyl- Alpha-methyl benzyl amine and 1 (P-tolyl) ethylamine. The method of claim 7, wherein said step (i) to provide a compound of formula III,
(a) providing a racemic mixture of a compound of formula (IX) with a compound of formula (X)
<Formula IX>

(X)

Wherein PG ⁶ and PG ⁷ are the same and each is a hydroxyl protecting group;
(b) reacting the mixture as defined in step (a) with a stereoselective enzyme that affects ester hydrolysis of the compound of formula IX to obtain a compound of formula III
Method comprising a. The method of claim 11, wherein the stereoselective enzyme is selected from Candida antarctica lipase B, porcine liver esterase or porcine pancreatic lipase. A method of making a PET tracer as defined in claim 1 comprising reacting a precursor compound of formula (I) as defined in claim 2 with a suitable source of ¹⁸ F. 6. The process of claim 13, wherein R ¹ is a leaving group for the precursor compound of Formula I and the suitable source of ¹⁸ F comprises ¹⁸ F-fluoride and a cationic counterion. 15. The method of claim 14, wherein the cationic counterion is selected from rubidium, cesium, potassium and tetraalkylammonium salts complexed with kryptand. 16. The method of any of claims 13-15, wherein the method is automated. i) a container containing a precursor compound as defined in any of claims 2 to 4; And
ii) means for eluting the vessel of step (i) with a suitable source of ¹⁸ F as defined in any of claims 13-15.
A cassette for performing the method as defined in claim 16 comprising a. ^{18. The} cassette of claim 17, further comprising (iii) an ion-exchange cartridge for removal of excess ¹⁸ F. A radiopharmaceutical composition comprising a PET tracer as defined in claim 1 together with a biocompatible carrier suitable for mammalian administration. i) administering a PET tracer to the subject as defined in claim 1;
ii) allowing the PET tracer to bind to PBR in the subject;
iii) detecting a signal emitted by ¹⁸ F included in the combined PET tracer;
iv) generating an image representing the location and / or amount of the signal; And
v) measuring the distribution and extent of PBR expression directly correlated with the signal in the subject
PET imaging method for measuring the distribution and / or extent of PBR expression in the subject. The method of claim 20, wherein the PET tracer is administered as a radiopharmaceutical composition as defined in claim 19. The method of claim 20 or 21, wherein the PET imaging method is performed repeatedly during the course of a treatment regimen for the subject comprising administration of a drug to combat PBR status. Diagnosis of a condition in which PBR is upregulated, comprising a PET imaging method as defined in claim 20 or 21, with an additional step (vi) of determining the distribution and extent of PBR expression as a result of a particular clinical picture. Way.

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